1. Field of the Invention
This invention relates to apparatus and methods for micro-organism culture and testing. In particular embodiments it relates to devices and methods suitable for application to motile bacteria such as species, strains, and serotypes of Salmonella, Campylobacter, Listeria, Vibrio, Aeromonas, or Escherichia.
These examples of motile bacteria are particularly important subjects of culture and testing methods adapted to the surveillance of food hygiene, environmental hygiene and pollution, arid clinical samples, owing to their pathogenic nature in humans and animals.
2. Description of Related Art
Current methods are protracted and laborious, especially for example those methods often used to test for Salmonella contamination in food samples.
One standard method of testing for Salmonella contamination, e.g. in a food sample, involves the steps or:
(a) dispersing a 25 g sample of food or other material to be tested in 225 ml of a `pre-enrichment` or `resuscitation` medium comprising for example bacteriological peptone, and incubating for up to one day (e.g. at 37 deg. C. for 18-24 hours): PA1 (b) subculturing the initial culture into each one of one or more different formulations (for example two) of `selective enrichment` medium to depress the growth of other micro-organisms relative to that of any Salmonella present, and growing these enrichment cultures by incubation to yield a demonstrable population of Salmonella (if present): (the least demonstrable population level can be very variable, but can be for example about 1000 organisms per ml in the absence of other contaminants, otherwise maybe up to about a million organisms per ml or more in the presence of heavy contamination with other organisms): PA1 (c) using the enriched cultures obtained to provide inocula for a series of selective differential solid growth media, usually on agar, in dishes allowing the identification of colonies of Salmonella if present in the original sample: PA1 (d) conducting at least biochemical and/or immunological (agglutination) tests on any colonies provisionally identified as Salmonella to confirm or contradict the identification. PA1 (a) preparing for bacteriological incubation a sample of material suspected of containing motile bacteria, PA1 (b) placing said material into a culture vessel containing liquid nutrient medium suitable for resuscitation and growth of motile bacteria, PA1 (c) providing a carrier partly dipping into said liquid nutrient medium but projecting above a surface of said liquid nutrient medium, said carrier containing supported nutrient medium selected from media suitable for bacteriologically selective growth and indication of motile bacteria, said carrier having an opening below said surface of said liquid nutrient medium for contact between said liquid medium in said vessel and said supported medium in said carrier, thereby to allow migration of said motile bacteria from said liquid into said supported medium during the culture, and PA1 (d) incubating said sample and said nutrient media to allow growth of any motile bacteria present in said sample in said liquid medium, and to allow migration of said motile bacteria during the culture into said supported medium and growth of said motile bacteria therein. PA1 (a) a culture vessel containing liquid nutrient medium suitable for resuscitation and growth of motile bacteria, and PA1 (b) a carrier partly dipping into said liquid nutrient medium and projecting above surface of said liquid nutrient medium, said carrier containing supported nutrient medium selected from media suitable for bacteriologically selective growth and indication of motile bacteria, said carrier having an opening below said surface of said liquid nutrient medium for contact between said liquid medium in said vessel and said supported medium in said carrier, thereby to allow migration of said motile bacteria from said liquid into said supported medium during culture of said bacteria. PA1 (a) Medium 1 (medium suitable for the resuscitation and growth of salmonellae in an incubation tub) can usefully be made up in aliquots of dry powder sufficient when taken up in sterile water to yield 225 ml of the following composition: PA1 (b) Medium 2 (LID) (selective supported medium with indicator for growth and transfer of motile Salmonella) comprises a dry powder of a composition suitable to yield on rehydration a highly viscous medium of the following composition marked "Wet": PA1 (c) Medium 3 (modified RV: based on Rappaport-Vassiliadis Medium) is an alternative selective supported medium with indicator, for growth and transfer of motile salmonellae) comprises a dry powder suitable to yield on rehydration a highly viscous medium of the following composition "Wet": PA1 (d) Medium 4 (BGD) (brilliant green desoxycholate indicator medium): PA1 (e) Medium 5 (LICNR) (`lysine iron cystine neutral red` indicator medium):
The whole process can take about a week.
Recently, improved immunological test methods have been developed which can substitute for parts (c) and (d) of the standard procedure and somewhat shorten the overall time taken.
The prior art also includes a technique proposed for the isolation of Salmonella by a selective motility system (P F Stuart and H Pivnick (1965), Applied Microbiology 13(3) pp 365-372). This involved the use of `semisolid` gel media contained in U-tubes, from one side-arm of which bacteria were to be harvested after inoculation at the other side-arm using a sample from a pre-enrichment culture as inoculum at the surface of the semisolid gel.
A further technique depending on the motility of the organisms to be detected is contained in U.S. Pat. No. 4,563,418 (Bio-Controls, Inc.). This describes a motility-immunoimmobilisation method for detection of motile organisms such as Salmonella, and discloses a motility vessel with an opening at each end, with a cad to cover each end opening, and containing a gelled motility medium. The system is used by placing antisera to immobilise Salmonella in contact with the gelled medium at one end, and placing a selective enrichment medium containing chemotactic attractant such as L-serine together with an inoculum of the organisms from a prior selective enrichment culture in contact with the gelled medium at the other end. A positive result after incubation is indicated by a band of immobilised bacteria near that end of the tube where the antisera were added.
A further review and description of Salmonella detection including the topic of motility enrichment has recently been published by J M De Smedt, R F Bolderdijk, H F Rappold and D Lautenschlaeger (1986) J. Food Protection vol 49 (7) pp 510-514, and mentions transferring inocula from pre-enrichment cultures to form spots on the surface of semisolid agar containing Rappaport-Vassiliadis broth: after 24 h incubation, samples from the edges of areas where migrated bacteria appeared to have grown were further subcultured and tested for presence of Salmonella.
U.S. Pat. No. 3,704,204 (Armour & Co) describes an attempt to achieve rapid testing for Salmonella, exemplified by incubating samples of meat and bone meal in nutrient media containing selective inhibitory agents, represented by bile salts, deoxycholate, lithium chloride and an acridine derivative (acriflavin), with a cotton swab saturated with lead acetate placed approximately an inch and a half above the surface of the medium: blackening of the swab after 24 h incubation was taken as a positive (suspicious) result. Numerous false positives were reported to be given by this method, though no false negatives.
In spite of these efforts, it still remains a problem, however, to shorten this process of testing for motile bacteria in a convenient way, particularly in respect of the earlier stages of culture also.